The mechanism of whey protein and blueberry juice mixed system fermented with Lactobacillus inhibiting Escherichia coli during storage

The aim of this study was to identify the antimicrobial effect and mechanism of whey protein and blueberry juice mixed systems fermented with Lactobacillus against Escherichia coli during storage. The whey protein and blueberry juice mixed systems were fermented with L. casei M54, L. plantarum 67, S. thermophiles 99 and L. bulgaricus 134 and had different antibacterial activities against E. coli during storage. The antimicrobial activity of the mixed whey protein and blueberry juice mixture systems was the highest, with an inhibition zone diameter of approximately 230 mm, compared with the whey protein or blueberry juice systems alone. There were no viable E. coli cells 7 h after treatment with of the whey protein and blueberry juice mixed systems as determined by survival curve analysis. Analysis of the inhibitory mechanism showed that the release of alkaline phosphatase, electrical conductivity, protein and pyruvic acid contents, and aspartic acid transaminase and alanine aminotransferase activity in E. coli increased. These results demonstrated that these mixed systems fermented with Lactobacillus, especially those containing blueberries, could inhibit the growth of E. coli and even cause cell death by destroying the cell membrane and cell wall.

on. The antimicrobial activity of these blueberry compounds may disrupt the integrity of the cell membranes of foodborne pathogens 8 . A study reported that probiotic-fermented blueberry juices improved the reducing power and showed stronger antibacterial activity against E. coli than fresh blueberry juice 9 . Another study noted that lactic acid bacteria have antibacterial effects due to the key antibacterial compounds they produce, such as bacteriocin, hydrogen peroxide and organic acids 10 . In addition, lactoferrin found in the whey protein fraction of milk can interact with the lipopolysaccharide layer of gram-negative bacteria, disrupt their outer membrane and enhance their susceptibility to the lysozyme by increasing membrane permeability 11 . However, the antibacterial effect and mechanism of a Lactobacillus-fermented whey protein and blueberry juice mixed system on foodborne pathogens is unclear. Thus, the inhibitory mechanisms of the mixed fermentation systems against foodborne pathogens during storage deserve deep investigation.
The objectives of this study were: (1) to examine the antibacterial effect of a Lactobacillus-fermented whey protein and blueberry juice mixed system on E. coli during storage, and (2) to investigate the possible antibacterial mechanism of this system from the perspective of cells membrane integrity. The Lactobacillus-fermented whey protein and blueberry juice mixed system showed high antibacterial activity (inhibition zone diameters and survival curves) against E. coli. Then, the cell membrane integrity (as determined by evaluating the activities of AKP, AST and ALT, electrical conductivity, and protein and pyruvate contents) was studied according to the inhibition mode. Therefore, the Lactobacillus-fermented whey protein and blueberry juice mixed system has potential for becoming a novel antibacterial agent and provides a basis for its application as an antibacterial agent in foods and medicines. Preparation of the blueberry and whey protein fermented beverages. The Lactobacillus strains were activated in 10 mL of Man-Rogosa-Sharpe (MRS) broth at 37 °C for 16 h. S. thermophiles was activated in 10 mL of M17 broth at 42 °C for 12 h. The mixed bacterial culture consisted of L. casei M54 and L. plantarum 67 at a ratio of 1:1, as well as S. thermophiles 99 and L. bulgaricus 134. The sample treatments of samples and Lactobacillus strains used were shown in Table 1. Blueberry juice was prepared from 17 g of blueberries by breaking the fruits with a crusher followed by filtering using a 200 mesh filter screen. Subsequently, the blueberry juice was mixed with distilled water to reach a blueberry juice content of 17% (w/v). One hundred millilitres of blueberry juice was added to 6 g (w/v) of sugar and 6 g (w/v) of whey protein powder. Then, the pH values of the blueberry and whey protein mixtures were adjusted to 6.5 and 7.0, and pasteurized in a water bath. After cooling to room temperature, 2 mL of the mixed bacterial culture was added to 100 mL of the blueberry and whey protein mixture to achieve an inoculum of 2% (v:v), respectively. The blueberry and whey protein mixtures fermented with L. casei M54 and L. plantarum 67 were incubated at 37 °C, and the other mixtures fermented with S. thermophiles 99 and L. bulgaricus 134 were incubated at 42 °C. www.nature.com/scientificreports/ Determination of the inhibition zone diameters. etermination of the E. coli inhibition zone diameters for the whey protein and blueberry juice mixed systems fermented with Lactobacillus was performed according to the method of Zhong et al. 9 with slight modification. Briefly, 100 μL of 1 × 10 6 CFU/mL E. coli suspensions was coated with glass coating rod uniformly on disposable sterilized solid agar plates. After 10 min, the medium was punched using a 10 mm diameter perforator that had been sterilized previously. Subsequently, 200 μL of the whey protein and blueberry juice mixtures fermented with Lactobacillus was placed into the holes with a pipette. Finally, the agar plates were placed in an electric 37 °C constant temperature incubator for 12 h.

Material and methods
Survival curve analysis. Survival curves were used to determine the mode of antibacterial action of the whey protein and blueberry juice mixed systems fermented with Lactobacillus against E. coli according to the method of Zhou et al. 8 . In brief, all whey protein and blueberry juice mixed systems fermented with Lactobacillus were added to E. coli suspensions (10 9 CFU/mL) to achieve a final concentration of 10 8 CFU/mL. E. coli incubated with LB medium was regarded as the control sample. Subsequently, the above mixture were incubated at 37 °C for 12 h, and the reduction in the number of viable calls was measured every two hours by the plate counting method.
Determination of the activity of AKP. The effects of the whey protein and blueberry juice mixed systems fermented with Lactobacillus on E. coli AKP activity were studied using an AKP assay kit according to the manufacturer's instructions. According to the method of Deng et al. 12 , the changes in absorbance at a wavelength of 520 nm represent the activity of AKP. Determination of the released protein content. The content of protein released from E. coli was determined using the BSA method. E. coli in the logarithmic phase were treated with the whey protein and blueberry juice mixed systems fermented with Lactobacillus for 10 h. The protein contents in the supernatants were assessed using the Pierce BCA Protein Kit (Thermo Scientific) purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) 14 .

Determination of the electrical conductivity.
Determination of the activity of AST and ALT. The activities of AST and ALT were determined as described previously for the determination of AKP activity. The activities of AST and ALT in the supernatants were assessed using the AST and ALT Kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions 15 .
Determination of the pyruvate content. The pyruvate content was determined from the measured AKP activity with some modifications. At first, the optical density of the supernatants at 600 nm was adjusted to 0.7. Then, the pyruvate content of E. coli was determined using a pyruvate kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions.
Statistical analysis. Collected data are expressed as the means ± standard deviations (SDs). Analysis of variance (ANOVA) was performed, and the means were compared by the Student-Newman-Keuls test. A value of p < 0.05 was considered to indicate statistical significance. Data were analysed by using a statistical software package (SPSS for Windows, 11.5,2002, SPSS Inc., USA).
Ethics approval and consent to participate. This experimental plant research (either cultivated or wild), including the collection of plant material, complied with institutional, national, or international guidelines. Field studies were conducted in accordance with local legislation. The plant materials have been widely consumed by local people and were used in our previous study.
Ethical approval. This article does not contain any studies with human or animal subjects.

Results and discussion
Antimicrobial properties. The antimicrobial properties of the whey protein and blueberry juice mixed systems fermented with Lactobacillus against E. coli during storage were determined by measuring the diameters of the zones of inhibition. As shown in Fig. 1, the antimicrobial activities of the blueberry and whey protein systems and the mixed systems fermented with L. casei M54 and L. plantarum 67 or S. thermophiles 99 and L. bulgaricus 134 were not significantly different. However, the blueberry and whey protein mixed fermentation systems showed the highest antimicrobial activity against E. coli after storage for 12 d, with inhibition zone diameters of approximately 230 mm and 225 mm, respectively. This result may be due to the increased release of polyphenols from the mixed blueberry and whey protein fermentation systems, as shown in our previous study 16 . It has been reported that polyphenols are the main antimicrobial agents affecting in vitro measurements 17  www.nature.com/scientificreports/ also suggested that the phenolics in blueberries are phytochemicals with strong antibacterial and antimycotic properties 9 . The antimicrobial activity of the blueberry and whey protein mixed system first increased and then decreased with prolonged storage time, which may be attributed to structural changes in the fermented antimicrobial products. In addition, the diameters of the antibacterial circles of the whey protein system fermented with S. thermophiles 99 and L. bulgaricus 134 against E. coli were 100 mm, 40 mm, 30 mm and 15 mm greater than those of the system fermented with L. casei M54 and L. plantarum 67, respectively. This effect may be closely related to the antimicrobial characteristics of lactic acid bacteria metabolites because different lactic acid bacteria have different metabolites including organic acids, fatty acids, short peptides and others 18 .

Effects of the mixed systems fermented with Lactobacillus on the survival of E. coli. To gain an
insight into the effects of the mixed systems fermented with Lactobacillus on the growth of E. coli, survival curve analysis was carried out. As depicted in Fig. 2, the untreated control E. coli cells grew steadily for 12 h to more than 9 log CFU in the initial stationary growth phase. Compared with the control, all systems fermented with Lactobacillus inhibited E. coli at the initial phase. With prolonged incubation time, the number of viable cells treated with the blueberry system increased. This result suggested that the blueberry system has bacteriostatic effects against E. coli 8 . In particular, the viable E. coli cell count after the bacteria were treated with the blueberry and whey protein mixed system fermented with L. casei M54 and L. plantarum 67, whey protein and the mixed system fermented with S. thermophiles 99 and L. bulgaricus 134 decreased from approximately 7 log CFU to 0 log CFU, which indicated that these fermentation systems have bacteriostatic and bactericidal effects on E. coli. The above results demonstrated these samples have the potential to become antibacterial agents against E. coli.

Effect of the mixed fermentation systems on the activity of AKP. Cell membrane destruction
has an unfavourable effect on the membrane-associated energy-transducing system. A study showed that the activity of AKP can regulate Ca 2+ metabolism, which is related to calcium binding protein, the associated ion channels and energy metabolism based on phosphate. Once AKP is inactivated, cell differentiation is prevented by controlling the progress of dephosphorylation 4 . The effect of the blueberry and whey protein mixed system fermented with Lactobacillus on the AKP activity in E. coli was represented in Fig. 3. AKP activity of E. coli treated with blueberry system was higher than that of blueberry and whey protein mixed system and whey pro- www.nature.com/scientificreports/ of the cell membrane increased after treatment with all of whey protein and blueberry juice mixed systems fermented with Lactobacillus, which resulted in the leakage of intracellular ions such as Na + and K + that play a vital role in maintaining the balance of the organism and adjusting metabolic activity 19 . The results were in agreement with those that found an increase in relative electric conductivity of E. coli O157:H7 treated with different concentrations of natural antimicrobials for 2, 6 and 24 h 14 . A similar study suggested that treatment with Rosa rugosa tea polyphenol increased the permeability of the S. aureus cell membrane and caused ion leakage, which caused an increase in the electrical conductivity in the culture medium and finally cell death 20 . After storage for 12 days, the electrical conductivity of all treated E. coli decreased significantly (p < 0.05), which may be related to the reduction in phenolic compound contents in the samples. Previous study showed that the blueberry polyphenol content in fermented yogurt gradually decreased after storage for 28 days 21   www.nature.com/scientificreports/ were relatively higher than those treated with S. thermophiles 99 and L. bulgaricus 134, which may be attributed to the production of different antibacterial substances. It was also suggested that the antibacterial substances of L. plantarum against E. coli are mainly organic acids and antimicrobial peptides 22 . The antibacterial effect of L. bulgaricus against E. coli has mainly been associated with lactic acid 23 .

Effects of the mixed fermentation systems on the content of protein released from E. coli. Whether the cell membrane is intact affects the growth and survival of bacterial cells. The bacterial cell
membrane is a selective permeability barrier to the internal and external environment that controls the penetration of nutrients to maintain normal bacterial growth and drain metabolic waste. Therefore, to investigate the cell membrane integrity of E. coli treated with the blueberry and whey protein mixed systems fermented with Lactobacillus, the content of cellular proteins leaked from E. coli was studied. Protein release from all samples remained stable, as shown in Fig. 5. The above results showed that the blueberry and whey protein mixed systems fermented with Lactobacillus have a significant inhibitory effect on E. www.nature.com/scientificreports/ coli. The content of leaked protein from the samples treated with the fermentation systems supplemented with blueberries were almost 33.33% higher than the contents leaked from the samples treated with the whey protein systems fermented with L. casei M54, L. plantarum 67 and S. thermophiles 99, L. bulgaricus 134 at 0 d. This result indicated that the main antibacterial compound may be associated with the components of blueberries. For example, the anthocyanin plus proanthocyanidin fraction plays a major role in damaging the integrity of the E. coli cell membrane 8 .

Effects of the mixed fermentation systems on AST and ALT concentrations in E. coli. AST
and ALT are important intracellular enzymes in the metabolic pathways of organisms, participating in amino acid metabolism. With increasing cell wall and cell membrane permeability, intracellular enzymes can leak out. The concentrations of AST and ALT were measured to determine the integrality of cell wall and cell membrane, as shown in Fig. 6. The AST concentration was not significantly different among the blueberry systems, whey protein and blueberry systems. Nevertheless, the concentrations of AST in the bacterial cultures treated with the whey protein system were relatively lower during storage. Besides, the AST concentrations in E. coli treated with the whey protein system fermented with L. casei M54 and L. plantarum 67 were 20.83%, 142.86%, and 27.03% higher than those treated with the www.nature.com/scientificreports/ after 8 days of storage. The ALT concentration results were roughly in agreement with those above. These results may be because antibacterial compounds such as bacteriocins that are produced by some Lactobacillus strains are more active at acidic pH than at neutral pH, which may have caused some of the antibacterial compounds to become inactive 24 .
Effects of the mixed fermentation systems on the pyruvate content. Pyruvate plays an essential role in the TCA cycle (tricarboxylic acid cycle) and glycolysis process, participating in the energy metabolism pathways in living organisms 25 . Therefore, the pyruvate content was measured to determine whether the cell membrane retained its integrity. As shown in Fig. 7, the pyruvate contents in E. coli cultures treated with systems fermented with L. casei M54 and L. plantarum 67 were approximately 54%, 140% and 50% higher than those in the systems fermented with S. thermophiles 99 and L. bulgaricus 134 at 0 d, respectively. Nevertheless, there were no significant differences after 12 days of storage. The results showed that the mixed systems fermented with L. casei M54 and L. plantarum 67 can disrupt E. coli membrane integrity, increase the permeability of the cell membrane and cause the leakage of pyruvate, affecting bacterial energy metabolism and finally leading to cell death. This may be related to the antimicrobial properties of probiotics. The result may be connected with the fact that the organic acids and fatty acids metabolized by L. plantarum, such as lactic acid and acetic acid, have strong antibacterial activity and can destroy the cell membrane integrity of E. coli and result in the leakage of its cellular contents, finally affecting normal bacterial growth 18 .

Conclusion
According to the above results, the fermentation systems that included blueberries were found to have the greatest antimicrobial effect against E. coli. For example, it was suggested that a whey protein and blueberry juice mixed system fermented with Lactobacillus displayed remarkable antibacterial characteristics against E. coli with antibacterial zone diameters of 230 nm and 225 mm at the end of storage, respectively. In particular, the blueberry systems had strong antibacterial effects against E. coli, as they disrupted the cell membrane integrity and affected the total content leakage, AKP activity, electrical conductivity and protein content. Additionally, the blueberry systems fermented with L. casei M54 and L. plantarum 67 also increased the pyruvate leakage, which interrupted normal growth of E. coli cells. Thus, the blueberry and whey protein mixed system fermented with Lactobacillus has great potential for future development as a natural antimicrobial agent to control pathogens. However, further studies are needed to explore what specific active components of the mixed systems fermented with Lactobacillus play a significant role in inhibiting the growth of E. coli and to investigate these antibacterial components against more microorganism strains. Furthermore, the results also indicated that the whey protein and blueberry juice mixed system fermented with Lactobacillus may become a promising natural preservative to prevent and control foodborne pathogens such as E. coli.